赤眼鳟β-肌动蛋白基因克隆、组织表达与稳定性分析

Cloning,expression and stability analysis ofβ-actin gene in squaliobarbus curriculus

为探究赤眼鳟(Squaliobarbus curriculus)β-肌动蛋白(β-actin)基因的结构和表达特性,本文采用RT-PCR技术和RACE法,从赤眼鳟肝脏中获得了赤眼鳟β-actin cDNA全长序列(GenBank:MT119965),该基因cDNA全长共1816 bp,由94 bp的5’端非编码区,594 bp的3’端非编码区和1128 bp的开放阅读框(95-1222)组成,阅读框共编码375个氨基酸。通过序列比对和系统进化树的构建,赤眼鳟β-actin与鲤科鱼类聚为一支且与鲮鱼、鲫鱼、黄颡鱼等多个物种的β-actin氨基酸同源性都为99%。荧光定量(qPCR)表达分析显示,β-actin在赤眼鳟肌肉、肠、脾脏、皮肤、血液、鳃、体肾、头肾、心脏和肝脏十个组织中都有表达,但不同组织的表达量存在差异。利用Best keeper软件对赤眼鳟体内的延伸因子1(elongation factor 1,EF1)和β-actin基因进行稳定性对比,发现β-actin基因稳定性低于EF1基因。以上结果为赤眼鳟的分子系统进化研究提供证据,也为赤眼鳟功能基因表达研究提供参考依据。

To investigate the structure and expression characteristics of beta-actin(β-actin)gene in Squaliobarbus curriculus,the full length sequence of Squaliobarbus curriculusβ-actin cDNA is cloned from the liver by the way of RT-PCR and RACE(GenBank:MT119965).The full length is 1816 bp,which consists of a 94 bp 5’untranslated region(UTR),a 594 bp 3’UTR and an 1128 bp open reading frame(95-1222).And there are 375 amino acids in the translated protein.The phylogenetic tree shows thatβ-actin of Squaliobarbus curriculus gathers into one branch with Cyprinidae.And sequence analysis reveals that the homology ofβ-actin amino acids with Cirrhinus molitorella,Carassius auratus and Tachysurus fulvidraco and other species is 99%,which suggestesβ-actin gene is highly conserved in fish.The expression analysis of qPCR shows thatβ-actin is expressed in 10 tissues including muscle,intestine,spleen,skin,blood,gill,body kidney,head kidney,heart and liver,but the expression level ofβ-actin is different among these tissues.The stability of the EF1(elongation factor 1)andβ-actin in Squaliobarbus curriculus is compared by the Best Keeper software.EF1 gene is more stable.The above research provides evidence for the evolution of the molecular system and a reference for the study of the gene expression of Squaliobarbus curriculus.